in the lungs and cranium of a mouse. The three x-ray sources with 1.5 mm focal spot combine to achieve an output of 200cGy/min at the 25 cm isocenter in air. For small field irradiation, the cylindrical Osmic lens accepts a 4 cm diameter x-ray beamat its entrance face, and emits a focused 40–80 keV pencil beam at the focal length 15 to 25 cm downstream. The selectionof 40–80 keV x-rays from the input spectrum is characteristics of the graphite lens. Using the 0.4 mm focal spot, the full widthhalf maximum of the pencil beam is 1.5 mm. The lens replacement gain factor is �3x, defined as the ratio of the output at focuswith and without the lens. Thus, 3 source/lens arrangement achieves a dose rate of �240 cGy/min and will compensate for theinherent inefficiency of pencil beam delivery. Each source/lens unit can also be pivoted on the gantry to enhance dose paintingcapability. At kV energies, EGSnrc calculations achieve about 5% agreement with relative dose measurements. The calculationsalso show that the 40–80 keV x-rays will be strongly attenuated by bone and renders the pencil beam more suitable forirradiation of mice or superficial structures. Finally, an optical CT system is used to measure 10,000 point doses at better than1 mm resolution in gel in �10 min, and will be most instrumental for commissioning the SARRP.

Conclusions: Small animals are invaluable to cancer research, in terms of cost and well-established tumor model systems.Performance of the component parts demonstrates that the SARRP can achieve highly localized irradiation of small animals.The new capabilities allow the important and timely study of tumor and normal tissue response to non-uniform dosedistributions, and novel treatment methods that combine conformal irradiation and other therapeutic agents, such as drugs,angiogenic modifiers and inducible gene products.

Supported in part by NCI grant R01CA108449

2012 Low-Level Radiation-Induced DNA Damage Evades Early Cellular Response Mechanisms Leading toIncreased Cell Death

S. Collis,1 J. Schwaninger,1 A. Ntambi,1 T. Keller,1 L. Dillehay,1 W. Nelson,1T. DeWeese1

1Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University, Baltimore, MD

Purpose/Objective: DNA damage that is not detected and repaired can lead to an increased frequency of chromosomalaberrations, a major contributing factor in the onset of tumorogenesis. Such unrepaired DNA damage can also lead to anincrease in mitotic cell death as the cell struggles to successfully segregate its chromosomes. We were interested in investigatingwhether radiation-induced DNA damage could be introduced into both normal and cancerous human cells at a level andfrequency that could evade detection by cellular repair mechanisms, ultimately resulting in increased cell death. We were alsointerested in studying the molecular mechanisms involved in this processes.

Materials/Methods: Multiple human cancer cell lines (HCT116, RKO, DU145 and PC-3) were exposed to either high (acute;�4500 cGy/hr) or low dose rate (LDR; 2 or 9.4 cGy/hr) ionizing radiation. Clonogenic survival assays, SDS/PAGE andimmunoblot analyses for activated ATM and FACS analyses for activated H2AX were performed following radiation exposureat both acute and LDR radiation exposures.

Results: We observed an increase in cell death among four different human cancer cell lines following LDR exposurescompared to acute exposures of equivalent dose. We demonstrated that this increased cell killing was a consequence ofineffective activation of the critical damage sensor ATM and its downstream target H2AX (a cellular marker of sites of DNAdamage). This phenomenon was also observed in normal primary human fibroblast cultures. The apparent reduction inclonogenic survival was not due to accumulation of senescent cells during the protracted radiation exposures prior to plating.The failure of LDR treated cells to sufficiently activate ATM was, in fact, not due to the presence of dysfunctional ATM proteinfollowing LDR exposure as cells pre-treated with LDR radiation were shown to elicit a normal ATM response followingsubsequent acute exposures.

Conclusions: The data presented here are the first to demonstrate that low levels of DNA damage introduced by LDR radiationdo not activate the DNA damage sensor ATM, a critical cellular response mechanism. This occurs despite the appearance ofactivated H2AX, a marker of DNA double-strand breaks. This lack of ATM activation and lower activated H2AX ultimatelyresults in greater amounts of cell killing compared to equivalent doses of acute radiation exposures. These findings may aid thefurther understanding of the early cellular DNA damage response mechanisms and have broad-range implications for clinicaltreatment of solid tumors.

2013 Oncogenic Transcription Factor kB Activation After Therapeutic Doses of Radiation TransactivatesTelomerase Reverse Transcriptase Promoter Induction

M. Natarajan,1 S. Mohan,2 F. N. Roldan,1 T. S. Herman,1 C. R. Thomas1

1Radiation Oncology, U.T. Health Science Center, San Antonio, TX, 2Pathology, U.T. Health Science Center, San Antonio,TX

Purpose/Objective: The objective of this study is to determine whether radiation exposure triggers the nuclear factor kb andthat the activation of NF-kB is responsible for the telomerase reverse transcriptase promoter activation and telomerase enzymeactivity.

Materials/Methods: Primary aortic endothelial cells were used for the study as they are routinely exposed to radiation duringtherapy. Cells were exposed to low LET radiation (0, 1 & 2 Gy) using 137Cs source at a dose rate of 1.29 Gy/min.Electrophoretic mobility shift assay (EMSA) was carried out to determine the NF-kB DNA-binding activity. Transienttransfections assays were used to examine the NF-kB-dependent TERT promoter activation. Telomerase Repeat AmplificationProtocol (TRAP) was used to determine the telomerase enzyme activity. NF-kB activation was blocked by IkB alpha mutant(IkB alphaS32A/S36A) and the expression of IkB alpha levels was assayed by Western blot analysis.

Results: Our results indicate that following radiation exposure (1 & 2 Gy), an increase in telomerase activity occurs. Kineticsanalysis showed that the initial induction in telomerase activity occurs as early as 8 h post irradiation. Subsequent analysisrevealed that the increased telomerse activity is due to increased enzyme synthesis resulting from an increased transcription.Also, we observed an enhanced transcription of the telomerase gene following 2 Gy. We further examined whether the

S347Proceedings of the 46th Annual ASTRO Meeting

induction, at least in part, mediated through the transcription factor NF-kB. Following gamma-radiation, NF-kB becomesfunctionally activated as an increase in its transactivating property occurs. More significantly, the 2 Gy-induced transcriptionalactivation of telomerase gene was abrogated by ectopically expressing the IkB alpha mutant (IkB alphaS32A/S36A), whichblocks NF-kB activation. Furthermore, deleting the NF-kB recognition site on telomerase promoter strongly attenuates the levelof transcriptional induction following radiation. Consistent with the notion that NF-kB mediates gamma-ray-induced telomeraseresponse, ectopically expressed Ik B alphaS32A/S36A also attenuated the ability of gamma ray to induce telomerase enzymeactivity.

Conclusions: Activation of NF-kB following low LET ionizing radiation exposure at doses used in therapeutic fractions, bytransactivating the TERT promoter, induces an increased transcription of telomerase reverse transcriptase and telomeraseenzyme expression. Increased telomerase in these cells may impart survival advantage. As radiotherapy plays a major role inthe management of multiple human cancers, understanding of the mechanism through which tumor cells acquire radio-resistance and survival advantage may provide an inroad to developing alternate therapeutic strategies.

2014 Modulating Radiation Response with the Histone Deacetylase (HDAC) Inhibitor SAHA in HumanCarcinomas

P. Chinnaiyan,1 G. Vallabhaneni,1 E. Armstrong,1 S. Huang,1 P. Harari1

1Radiation Oncology, University of Wisconsin, Madison, WI

Purpose/Objective: A broad series of molecularly targeted anti- cancer agents are advancing into clinical trials. Several ofthese targeted agents hold promise as radiation sensitizers based on their capacity of inhibiting pro-survival signals whichpotentially confer radiation resistance to cancer cells. Histone deacetylase (HDAC) inhibitors, which modulate chromatinstructure and gene expression, represent a specific class of anti-cancer agents which hold particular potential as radiationsensitizers. In this study, we examine the capacity of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) to modulateradiation response in human carcinoma cell lines and explore potential mechanisms underlying these interactions.

Materials/Methods: Cell Proliferation: Exponentially growing tumor cells were incubated in medium containing 0–10 uM ofSAHA for 72 hours. Cells were fixed/stained with crystal violet and the absorbance was read to determine cell viability.

Apoptosis: Caspase activity was analyzed by fluorescence spectroscopy using a fluorescein labeled pan-caspase inhibitor.Cells were harvested after 48 hours exposure to SAHA (1.0 uM), radiation (6 Gy), or the combination. Whole cell lysates wereevaluated for PARP cleavage by western blot analysis.

Radiation Survival: Cells were exposed to varying doses of radiation � 3 day pre-treatment with SAHA (0.75–1.0 uM).Following incubation intervals of 14–21 days, colonies were stained with crystal violet and manually counted.

Immunocytochemistry: Cells were grown and treated in chamber slides. At specified times after treatment with SAHA, cellswere fixed, permeabilized, and probed with primary and secondary antibody solutions. Slides were analyzed using anepifluorescent microscope.

Results: SAHA induced a dose dependent inhibition of proliferation in human prostate (DU145) and glioma (U373vIII) cancercell lines. Anti-proliferative effects were markedly enhanced when evaluated for the ability to form viable clonogens followingexposure to SAHA. Exposure to SAHA induced a supra-additive enhancement of apoptosis when combined with radiation asmeasured by caspase activity (p � 0.05) and PARP cleavage. The impact of SAHA on radiation response was furthercharacterized using clonogenic survival analysis, which demonstrated that treatment with SAHA reduced tumor survivalfollowing radiation exposure. We identified several oncoproteins and DNA damage repair proteins that are differentiallyregulated by SAHA, which may contribute to mechanistic synergy between HDAC inhibition and radiation response. Theseproteins include EGFR, AKT, DNA-PK, and Rad51. Fluorescent immunocytochemistry following 24 hr exposure to SAHAdemonstrated striking changes in cellular morphology and perinucleur localization/degradation of the EGFR.

Conclusions: These pre-clinical results indicate that treatment with the HDAC inhibitor SAHA can enhance radiation-inducedcytoxicity in human tumor cells. We are now examining the capacity of HDAC inhibitors to modulate radiation response andtumor control in animal xenograft model systems to strengthen the rationale for future clinical trial exploration.

2015 Novel Use of Zebrafish as a Vertebrate Model to Screen Radiation Protectors and Sensitizers

A. P. Dicker,1 M. F. McAleer,1 E. Santana,3 S. A. Farber,3 U. Rodeck2

1Radiation Oncology, Thomas Jefferson University, Philadelphia, PA, 2Dermatology and Cutaneous Biology, ThomasJefferson University, Philadelphia, PA, 3Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA

Purpose/Objective: Zebrafish (Danio rerio) embryos provide a unique vertebrate model to screen therapeutic agents, becauseof their closer genetic relationship to humans than yeast or insects, ready abundance and accessibility, and optical transparencythroughout their brief development. We have evaluated the effects of ionizing radiation and screened known radiation modifiersusing zebrafish embryos.

Materials/Methods: Wildtype zebrafish adults were mated, and viable embryos collected and plated for exposure to doses of0–10 Gy single-fraction 250 kVp X-rays in the presence or absence of either the radioprotective thiophosphate Amifostine (0–4mM) or the radiosensitizing EGFR inhibitor AG1478 (0–10 �M) at various stages of embryonic development from 1–24 hourspost fertilization (hpf). Dechorionated embryos were examined for morphologic abnormalities and viability up to 144 hpf.

Results: Ionizing radiation alone produced a time-and dose-dependent perturbation of normal development and survival ofexposed embryos with maximal sensitivity detected at doses �4 Gy delivered prior to midblastula transition (4 hpf). This effectwas attenuated by pre-incubation with Amifostine, while pretreatment with AG1478 enhanced teratogenicity and lethality,particularly at therapeutically relevant (2–4 Gy) doses of radiation.

S348 I. J. Radiation Oncology ● Biology ● Physics Volume 60, Number 1, Supplement, 2004